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Toscana virus ( TOSV), West Nile virus ( WNV) and tickborne encephalitis virus ( TBEV) are among major viral pathogens causing febrile disease and meningitis/encephalitis. The impact of these viruses was investigated at a referral centre in Ankara Province, Central Anatolia in 2012, where previous reports suggested virus circulation but with scarce information on clinical cases and vector activity. Serum and/or cerebrospinal fluid samples from 94 individuals were evaluated, in addition to field-collected arthropod specimens that included 767 sandflies and 239 mosquitoes. Viral nucleic acids in clinical samples and arthropods were sought via specific and generic nested/real-time PCRs, and antibody responses in clinical samples were investigated via commercial indirect immunofluorescence tests ( IIFTs) and virus neutralization. A WNV antigen assay was also employed for mosquitoes. WNV neuroinvasive disease has been identified in a 63-year-old male via RNA detection, and the WNV strain was characterized as lineage 1. TOSV infections were diagnosed in six individuals (6.3%) via RNA or IgM detection. Partial sequences in a 23-year-old female, presented with fever and transient pancytopenia, were characterized as TOSV genotype A. Febrile disease with arthralgia and/or peripheral cranial nerve involvement was noted in cases with TOSV infections. Previous WNV and TOSV exposures have been observed in 5.3% and 2.1% of the subjects, respectively. No confirmed TBEV exposure could be identified. Morphological identification of the field-collected mosquitoes revealed Culex pipiens sensu lato (74.4%), Anopheles maculipennis (20.9%), An. claviger (2.1%) and others. Sandfly species were determined as Phlebotomus papatasi (36.2%), P. halepensis (27.3%), P. major s. l. (19.3%), P. sergenti (8.9%), P. perfiliewi (4.4%), P. simici (2.6%) and others. Viral infections in arthropods could not be demonstrated. TOSV genotype A and WNV lineage 1 activity have been demonstrated as well as serologically proven exposure in patients. Presence of sandfly and mosquito species capable of virus transmission has also been revealed. [ABSTRACT FROM AUTHOR]

Summary Toscana virus (TOSV), a sandfly fever virus serotype of medical and public health importance, is a major pathogen involved in aseptic meningtis occurring in Mediterranean countries and poses a threat to the residents as well as travellers. Limited data on TOSV activity are present from Turkey despite being located in the endemic zone. We aimed to identify TOSV exposure in 1115 healthy blood donors at the Hacettepe University Hospital Blood Bank in Ankara, Turkey, using commercial indirect fluorescence assays (IFAs) and virus neutralization test (VNT) for antibody detection and specificity confirmation. A total of 199 samples (17.8%) were positive for anti-TOSV that include IgG reactivity in 10.4%, IgM reactivity in 8.2% and IgM + IgG reactivity in 0.7% of the sera. Anti-TOSV specificity could be confirmed via VNT in 56% of the IgG- and 43.6% of the IgM-positive sera, making up a total of 58 samples (5.2%). Risk factors associated with TOSV IgG reactivity were male gender, residing in rural areas, frequent sighting of mosquitoes/sandflies and working outdoors. TOSV-specific antibody prevalence increased significantly with age. Evidence of exposure to other sandfly fever viruses was noted. These data reveal that mild or asymptomatic infections with TOSV are frequent in central and northern Anatolia. TOSV exposure has also been identified in residents of 9 provinces in southern/southeastern Anatolia for the first time. [ABSTRACT FROM AUTHOR]

Triatomines are infamous as vectors of the parasite Trypanosoma cruzi, the causative agent of Chagas disease. However, climate-driven range expansion and urbanization adaptation of triatomine populations, coupled with their highly diverse feeding strategies (vertebrate haematophagy, kleptohaematophagy, and coprophagy), and has elevated interest in triatomines as potential arboviral vectors. Information on the triatomine virome is scant, with prior records including only eight insect-specific viruses: Triatoma virus (TrV) and Rhodnius prolixus viruses 1-7. Here, we leverage publicly available transcriptome datasets to assess viral diversity in 122 wild and colony kissing bugs representing eight species from six countries. In total, six viruses were detected (including Rhodnius prolixus viruses 4-6), and TrV was detected in almost half of all screened triatomines. This is the first report of TrV in Triatoma brasiliensis and in members of the genus Mepraia (M. gajardoi, M. spinolai, and M. parapatrica), and this effort has vastly expanded the publicly available genomic resources of TrV, adding 39 genome sequences to the single genome sequence currently available in the GenBank database. Furthermore, two additional viruses-Meccus longipennis virus 1 and Drosophila melanogaster Nora virus-are herein reported for the first time from kissing bugs. Meccus longipennis virus 1 was detected in Triatoma infestans from Argentina, Brazil, Chile, and Peru, and Drosophila melanogaster Nora virus was found in T. infestans from Argentina. Our results illustrate the advantage and utility of low-cost transcriptome data mining for the discovery of known and novel arboviruses in triatomines and other potential insect vectors., (© 2024. The Author(s).)

Members of the Orthonairovirus genus (family Nairoviridae) include many tick-borne viruses of significant human and animal health impact, with several recently-documented pathogenic viruses lacking sufficient epidemiological information. We screened 215 adult ticks of seven species collected in Bulgaria, Georgia, Latvia and Poland for orthonairoviruses, followed by nanopore sequencing (NS) for genome characterization. Initial generic amplification revealed Sulina virus (SULV, Orthonairovirus sulinaense), for which an updated amplification assay was used, revealing an overall prevalence of 2.7% in Ixodes ricinus ticks from Latvia. Three complete and additional partial SULV genomes were generated, that consistently formed a separate, distinct clade with further intragroup divergence in the maximum likelihood analyses. Comparisons with previously described viruses from Romania exhibited similar genome topologies, albeit with divergent motifs and cleavage sites on the glycoprotein precursor. Preliminary evidence of recombination involving the S segment was documented, in addition to variations in predicted viral glycoproteins. Generic screening further identified Tacheng tick virus 1 (TCTV1, Orthonairovirus tachengense), with documented human infections, in Dermacentor reticulatus ticks from Poland, with a prevalence of 0.9%. Subsequent NS and assembly provided the first complete TCTV1 genome outside of China, where it was originally described. Phylogenetic analysis of virus genome segments revealed TCTV1-Poland as a discrete taxon within the TCTV1 cluster in the Orthonairovirus genus, representing a geographically segregated clade. Comparable genome topology with TCTV1 from China was observed, aside from minor variations in the M segment. Similar to SULV, TCTV1 exhibited several mismatches on previously described screening primer binding sites, likely to prevent amplification. These findings indicate presence of novel TCTV1 and SULV clades in Eastern Europe, confirming the expansion of orthonairoviruses with pathogenic potential., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The threat posed by emerging infectious diseases is a major concern for global public health, animal health and food security, and the role of birds in transmission is increasingly under scrutiny. Each year, millions of mass-reared game-farm birds are released into the wild, presenting a unique and a poorly understood risk to wild and susceptible bird populations, and to human health. In particular, the shedding of enteric pathogens through excrement into bodies of water at shared migratory stop-over sites, and breeding and wintering grounds, could facilitate multi-species long-distance pathogen dispersal and infection of high numbers of naive endemic birds annually. The Mallard ( Anas platyrhynchos ) is the most abundant of all duck species, migratory across much of its range, and an important game species for pen-rearing and release. Major recent population declines along the US Atlantic coast has been attributed to game-farm and wild mallard interbreeding and the introduction maladaptive traits into wild populations. However, pathogen transmission and zoonosis among game-farms Mallard may also impact these populations, as well as wildlife and human health. Here, we screened 16 game-farm Mallard from Wisconsin, United States, for enteric viral pathogens using metatranscriptomic data. Four families of viral pathogens were identified - Picobirnaviridae (Genogroup I), Caliciviridae (Duck Nacovirus ), Picornaviridae (Duck Aalivirus ) and Sedoreoviridae (Duck Rotavirus G). To our knowledge, this is the first report of Aalivirus in the Americas, and the first report of Calicivirus outside domestic chicken and turkey flocks in the United States. Our findings highlight the risk of viral pathogen spillover from peri-domestically reared game birds to naive wild bird populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bourke, Dusek, Ergunay, Linton and Drovetski.)

Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum., Competing Interests: Declaration of Competing Interest None, (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: This manuscript was prepared whilst KE and BPB held a National Research Council (NRC) Research Associateship Awards at the Walter Reed Biosystematics Unit (WRBU), through the Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, USA.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples., (© 2023. The Author(s).)

Introduction: We evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays., Methods: Forty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach., Results: Eleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted segment read counts was observed between PCR-positive and PCR-negative samples. NS further enabled the initial description of Quaranjavirus sequences in ticks, where human and avian pathogenicity of particular isolates had been previously documented., Discussion: NS was observed to surpass broad-range and nested amplification in detection and to generate sufficient genome-wide data for investigating virus diversity. It can be employed for monitoring pathogens in tick vectors or human/animal clinical samples in hot-spot regions for examining zoonotic spillover., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ergunay, Dincer, Justi, Bourke, Nelson, Liao, Timurkan, Oguz, Sahindokuyucu, Gokcecik, Reinbold-Wasson, Jiang, Achee, Grieco and Linton.)

Background and Methods: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing., Results: Viruses belonging to the Astroviridae , Circoviridae , Coronaviridae , Dicistroviridae , Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis , Myotis ricketti , Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified., Conclusions: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.

Blastocystis sp. and Dientamoeba fragilis are two most common protists worldwide, whose pathogenic potentials are a matter of debate since their discovery. This study aims to investigate the relationship between the activation of ulcerative colitis (UC) and irritable bowel syndrome (IBS) with these protists. A total of 100 patients (35 IBS, 35 active UC, and 30 remittent UC), diagnosed at Hacettepe University Adult Hospital (Ankara, Turkey), were screened for D. fragilis and Blastocystis sp. with microscopic examination using the methods of wet mount, trichrome staining, conventional PCR, nested PCR, real-time PCR and genotyping. Eight patients (4 IBS, 2 active, and 2 remittent UC patients) were found to be D. fragilis positive. 18S rRNA region of the parasite was amplified in four of the patients, whereas cathepsin L-like cysteine peptidase; clan Sc, family S9, serine peptidase; and clan MH, family M20 metallopeptidase in six different patients. All isolates were Genotype 1. Sequence results showed very limited diversity. A total of nine patients (3 IBS, 5 active UC, 1 remittent UC) were found to be positive for Blastocystis sp., all of which were Subtype 3. One active UC and one IBS patient were found to be positive for both parasites. No statistically significant difference was detected between the patient groups in means of parasite detection. D. fragilis was found to be related to older age (p=0,045). In our study, no significant correlation was identified between D. fragilis and Blastocystis sp., and the activation of UC and IBS. More studies are needed on the host-parasite relationship, including the role of gut microbiota, together with transcriptomic and metabolomic assessments to unveil the pathogenicity of both protists., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Focusing on the utility of ticks as xenosurveillance sentinels to expose circulating pathogens in Kenyan drylands, host-feeding ticks collected from wild ungulates [buffaloes, elephants, giraffes, hartebeest, impala, rhinoceros (black and white), zebras (Grévy's and plains)], carnivores (leopards, lions, spotted hyenas, wild dogs), as well as regular domestic and Boran cattle were screened for pathogens using metagenomics. A total of 75 host-feeding ticks [ Rhipicephalus (97.3%) and Amblyomma (2.7%)] collected from 15 vertebrate taxa were sequenced in 46 pools. Fifty-six pathogenic bacterial species were detected in 35 pools analyzed for pathogens and relative abundances of major phyla. The most frequently observed species was Escherichia coli (62.8%), followed by Proteus mirabilis (48.5%) and Coxiella burnetii (45.7%). Francisella tularemia and Jingmen tick virus (JMTV) were detected in 14.2 and 13% of the pools, respectively, in ticks collected from wild animals and cattle. This is one of the first reports of JMTV in Kenya, and phylogenetic reconstruction revealed significant divergence from previously known isolates and related viruses. Eight fungal species with human pathogenicity were detected in 5 pools (10.8%). The vector-borne filarial pathogens ( Brugia malayi, Dirofilaria immitis, Loa loa ), protozoa ( Plasmodium spp., Trypanosoma cruzi ), and environmental and water-/food-borne pathogens ( Entamoeba histolytica, Encephalitozoon intestinalis, Naegleria fowleri, Schistosoma spp., Toxoplasma gondii , and Trichinella spiralis ) were detected. Documented viruses included human mastadenovirus C, Epstein-Barr virus and bovine herpesvirus 5, Trinbago virus, and Guarapuava tymovirus-like virus 1. Our findings confirmed that host-feeding ticks are an efficient sentinel for xenosurveillance and demonstrate clear potential for wildlife-livestock-human pathogen transfer in the Kenyan landscape., Competing Interests: MM was employed by Kenya Wildlife Services Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ergunay, Mutinda, Bourke, Justi, Caicedo-Quiroga, Kamau, Mutura, Akunda, Cook, Gakuya, Omondi, Murray, Zimmerman and Linton.)

The host immunity is crucial in determining the clinical course and prognosis of coronavirus disease 2019, where some systemic and severe manifestations are associated with excessive or suboptimal responses. Several antigenic epitopes in spike, nucleocapsid and membrane proteins of severe acute respiratory syndrome coronavirus 2 are targeted by the immune system, and a robust response with innate and adaptive components develops in infected individuals. High titer neutralizing antibodies and a balanced T cell response appears to constitute the optimal immune response to severe acute respiratory syndrome coronavirus 2, where innate and mucosal defenses also contribute significantly. Following exposure, immunological memory seems to develop and be maintained for substantial periods. Here, we provide an overview of the main aspects in antiviral immunity involving innate and adaptive responses with insights into virus structure, individual variations pertaining to disease severity as well as long-term protective immunity expected to be attained by vaccination., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Context: Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder. Emerging animal and human data point to various changes in microbiota that could be linked with the syndrome. However, the effects of therapeutic approaches on gut microbial composition in women with PCOS remain unknown., Objective: We aimed to assess whether gut microbial composition is altered in PCOS and to determine the potential impact of oral contraceptive (OC) use on gut microbiota., Design: Prospective observational study., Setting: Tertiary referral hospital., Patients and Other Participants: The study included 17 overweight/obese patients with PCOS and 15 age- and body mass index-matched healthy control women., Main Outcome Measures: At baseline, clinical, hormonal, and metabolic evaluations and gut microbial composition assessment by 16S rRNA gene amplicon sequencing were performed for both groups. All measurements were repeated in patients after receiving an OC along with general lifestyle advice for 3 months., Results: Alpha and beta diversity did not show a difference between patients with PCOS and healthy controls at baseline and remained unaltered after 3 months of OC use in the PCOS group. Relative abundance of Ruminococcaceae was higher in PCOS (P = 0.006) and did not show a significant change after treatment., Conclusion: Women with PCOS have an increased abundance of Ruminococcaceae, whereas short-term OC use does not alter compositional features of gut microbiota in the syndrome., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: In an attempt to identify a wide spectrum of viral infections, cerebrospinal fluid (CSF) specimens were collected from pediatric cases with the preliminary diagnosis of viral encephalitis/meningoencephalitis in two reference hospitals, from October 2011 to December 2015., Methodology: A combination of nucleic acid-based assays, including in house generic polymerase chain reaction (PCR) assays for enteroviruses, flaviviruses and phleboviruses, a commercial real-time PCR assay for herpesviruses and a commercial real time multiplex PCR, enabling detection of frequently-observed viral, bacterial and fungal agents were employed for screening., Results: The microbial agent could be characterized in 10 (10%) of the 100 specimens. Viral etiology could be demonstrated in 7 (70%) specimens, which comprises Human Herpesvirus 6 (4/7), Herpes Simplex virus type1 (2/7) and Enteroviruses (1/7). In 3 specimens (30%), Streptococcus pneumoniae, Listeria monocytogenes and Staphylococcus aureus were detected via the multiplex PCR, which were also isolated in bacteriological media. All specimens with detectable viral nucleic acids, as well as unreactive specimens via nucleic acid testing remained negative in bacteriological cultures., Conclusions: Herpes and enteroviruses were identified as the primary causative agents of central nervous system infections in children. Enterovirus testing must be included in the diagnostic work-up of relevant cases., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2020 Aylin Altay-Kocak, Gulendam Bozdayi, Janine Michel , Meltem Polat, Saliha Kanik-Yuksek , Hasan Tezer, Aykut Ozkul, Kamruddin Ahmed, Andreas Nitsche, Koray Ergunay.)

Several health-promoting effects of kefir have been suggested, however, there is limited evidence for its potential effect on gut microbiota in metabolic syndrome This study aimed to investigate the effects of regular kefir consumption on gut microbiota composition, and their relation with the components of metabolic syndrome. In a parallel-group, randomized, controlled clinical trial setting, patients with metabolic syndrome were randomized to receive 180 mL/day kefir ( n = 12) or unfermented milk ( n = 10) for 12 weeks. Anthropometrical measurements, blood samples, blood pressure measurements, and fecal samples were taken at the beginning and end of the study. Fasting insulin, HOMA-IR, TNF-α, IFN-γ, and systolic and diastolic blood pressure showed a significant decrease by the intervention of kefir ( p ≤ 0.05, for each). However, no significant difference was obtained between the kefir and unfermented milk groups ( p > 0.05 for each). Gut microbiota analysis showed that regular kefir consumption resulted in a significant increase only in the relative abundance of Actinobacteria ( p = 0.023). No significant change in the relative abundance of Bacteroidetes, Proteobacteria or Verrucomicrobia by kefir consumption was obtained. Furthermore, the changes in the relative abundance of sub-phylum bacterial populations did not differ significantly between the groups ( p > 0.05, for each). Kefir supplementation had favorable effects on some of the metabolic syndrome parameters, however, further investigation is needed to understand its effect on gut microbiota composition.

Introduction: Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts for viruses, dissemination vehicles for the vectors, and sources of emerging strains in cross-species transmission. Turkey provides diverse habitats for a variety of wild birds and is located along major bird migration routes. This study was undertaken to provide a cross-sectional screening of avian specimens for a spectrum of vector-borne viruses., Materials and Methods: The specimens were collected in Hatay province, in the Mediterranean coast of the Anatolian peninsula, located in the convergence zone of the known migration routes. Generic PCR assays were used for the detection of members of Nairovirus, Flavivirus, and Phlebovirus genera of Flaviviridae and Bunyaviridae families. The circulating viruses were characterized via sequencing and selected specimens were inoculated onto Vero cell lines for virus isolation., Results and Discussion: Specimens from 72 wild birds belonging in 8 orders and 14 species were collected. A total of 158 specimens that comprise 32 sera (20.3%) from 7 species and 126 tissues (79.7%) from 14 species were screened. Eight specimens (8/158, 5%), obtained from 4 individuals (4/72, 5.5%), were positive. West Nile virus (WNV) lineage 1 sequences were characterized in the spleen, heart, and kidney tissues from a lesser spotted eagle (Clanga pomarina), which distinctly clustered from sequences previously identified in Turkey. Toscana virus (TOSV) genotype A and B sequences were identified in brain and kidney tissues from a greater flamingo (Phoenicopterus roseus), a great white pelican (Pelecanus onocrotalus), and a black stork (Ciconia nigra), without successful virus isolation. Partial amino acid sequences of the viral nucleocapsid protein revealed previously unreported substitutions. This study documents the involvement of avians in WNV dispersion in Anatolia as well in TOSV life cycle.

Sandfly-transmitted phleboviruses are globally spread agents causing febrile diseases and central nervous system infections. The activity of pathogenic phleboviruses, as well as several novel strains, has been reported from Turkey, a transboundary country connecting Asia, Europe, and Africa with suitable habitats for sandflies. This study overviews all published data on phleboviruses from Turkey and evaluates the impact from the virological, epidemiological, and public health perspectives. A systematic review of Web-based global and local resources was performed. Comparison and phylogenetic analyses of particular phlebovirus sequences were also undertaken. Through the evaluation of 1693 international and regional entries, 31 manuscripts providing data on case reports or outbreaks, serological surveillance, animal infections and exposure, virus characterization, vector surveillance, and/or diagnostics were accessed. Detailed information on 5 novel phleboviruses completely or partially characterized during 2008-2015 as well as on clinical and epidemiological features of major phleboviruses established as human pathogens such as Toscana virus and sandfly fever Sicilian virus has been compiled. The ongoing activity of these agents, as indicated by consistently reported symptomatic cases and confirmed exposure in vertebrates including humans, was noted. The circulation in the Anatolian peninsula of phleboviruses with surprising diversity as well as distinct virus species is documented. Specific phlebovirus strains constitute a public health threat for local populations and travelers and must be considered in the diagnostic workup of clinically compatible cases. Human health impact and epidemiological aspects of certain viruses require further investigation via intensive surveillance., (Copyright © 2016 John Wiley & Sons, Ltd.)

Several viral diseases are associated with altered microRNA (miRNA) expression, which can provide vital information about how cellular pathways respond to infection. However, the miRNA profile of Crimean Congo Hemorrhagic Fever (CCHFV) infections are not known. To address this gap, we performed real-time PCR-based miRNA analysis in individuals with acute Crimean Congo Hemorrhagic Fever (CCHFV) infections, with the goal of identifying pathways that might be associated with this disease. Peripheral blood mononuclear cells were analysed in eight individuals with detectable viral RNA and compared to five healthy subjects. A total of 106 differentially expressed miRNAs were identified, of which 19 miRNAs were either fivefold prominently up- or down-regulation. Several miRNAs associated with cytokine expression, some of which were previously associated with Dengue and Hantavirus infections were revealed. Moreover, possible mechanisms related to secretion of adhesion molecules and viral escape from innate immunity were identified. Pathway enrichment analyses further revealed the putative involvement of TNF-alfa, TGF-beta, MAPK, WNT, and neurotrophin signaling pathways in disease pathogenesis. J. Med. Virol. 89:417-422, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The purpose of this review is to provide practical information to help researchers intending to perform "from field to laboratory" studies on phleboviruses transmitted by sandflies. This guideline addresses the different steps to be considered starting from the field collection of sandflies to the laboratory techniques aiming at the detection, isolation, and characterization of sandfly-borne phleboviruses. In this guideline article, we address the impact of various types of data for an optimal organization of the field work intending to collect wildlife sandflies for subsequent virology studies. Analysis of different data sets should result in the geographic positioning of the trapping stations. The overall planning, the equipment and tools needed, the manpower to be deployed, and the logistics to be anticipated and set up should be organized according to the objectives of the field study for optimal efficiency.

In this series of review articles entitled "Practical guidelines for studies on sandfly-borne phleboviruses," the important points to be considered at the prefieldwork stage were addressed in part I, including parameters to be taken into account to define the geographic area for sand fly trapping and how to organize field collections. Here in part II, the following points have been addressed: (1) factors influencing the efficacy of trapping and the different types of traps with their respective advantages and drawbacks, (2) how to process the trapped sand flies in the field, and (3) how to process the sand flies in the virology laboratory. These chapters provide the necessary information for adopting the most appropriate procedures depending on the requirements of the study. In addition, practical information gathered through years of experience of translational projects is included to help newcomers to fieldwork studies.

Introduction: Toscana virus (TOSV) is a sandfly-borne bunyavirus with a significant public health impact. Preliminary studies have revealed TOSV exposure in dogs and they were suggested as potential reservoirs. This study was performed to characterize canine TOSV infections in an endemic region. Sequencing of TOSV small (S) segment in several previously identified specimens was also undertaken to reveal viral genealogy., Materials and Methods: Canine and feline plasma were collected in several districts of Mersin province, Mediterranean Anatolia, Turkey, during May-September, 2015. Phlebovirus RNA was screened through two nested polymerase chain reaction (PCR) assays, targeting S and large (L) segments of the viral genome. A kinetoplast minicircle nested PCR was employed for Leishmania DNA detection and typing. Previously collected TOSV-positive specimens from humans, dogs, cats, and sandflies from various regions in Turkey and Cyprus were further evaluated through the S segment PCR. All amplicons were characterized through sequencing., Results: A total of 210 specimens that comprise canine (76.2%) and feline (23.8%) plasma were screened. In three (1.9%) and two (1.3%) canine specimens, TOSV and Leishmania nucleic acids were detected, respectively. The TOSV strains were characterized as genotype B, and Leishmania infantum was identified in positive specimens. Twenty-four partial S segment sequences were amplified, which demonstrated a maximum intramural diversity of 3.88% in the nucleotide level. Sequence comparisons revealed significant similarities to particular genotype B strains characterized in Spain and France, whereas a notable divergence was observed among several TOSV strains. Single or recurrent amino acid substitutions were noted in eight residues of the viral nucleocapsid., Discussion: Canine infections of TOSV genotype B, with temporal and spatial association with L. infantum, were detected. Divergent TOSV S segment sequences with amino acid substitutions, presumably associated with host adaptation, were observed.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted arbovirus causing human disease in Europe and Asia. Over the past decades, the incidence of TBEV infection has significantly increased, with over 13,000 annual hospital referrals in endemic countries and cases emerging in previously unaffected regions. Specific detection of TBEV is required to diagnose suspected human cases or during surveillance of tick vectors and/or susceptible animal species. Widely used techniques for diagnosis comprise serological methods to detect viral antigens or antibodies and nucleic acid tests to detect viral RNA in target specimens. Moreover, virus isolation using susceptible cell lines or vertebrates, electron microscopy, or immunohistochemistry can also be employed on specific occasions. The purpose of this review is to compile and outline various approaches and techniques for detecting TBEV infection in ticks, wild animals, and humans. Specific sections for specimen collection and storage, nucleic acid testing, and serological assays cover various aspects of dynamics, performance characteristics, and utility in the diagnostic workup of suspected cases. Impact of immunoglobulin M testing and quantification, immunoglobulin G avidity, and real-time and quantitative polymerase chain reaction methods were overviewed with assay comparisons. Recent advances in serological assays to mitigate the impact of cross-reactions were further discussed along with the detailed interpretation of laboratory test results in human infections.

An increased incidence of chronic kidney disease (CKD) after West Nile Virus (WNV) infections has been suggested but the association of WNV infections with renal damage remain inconclusive. This study was undertaken to characterize WNV infections in individuals with acute kidney injury (AKI) and CKD, and to evaluate hemodialysis as a probable transmission route. A total of 463 plasma and urine samples were collected from 45 AKI and 77 CKD patients. Nested and real-time polymerase chain reaction (PCR) assays were employed for viral RNA detection. Specific immunoglobulins were investigated via immunofluorescence and plaque reduction neutralization assays. Consecutive pre and post-dialysis samples were evaluated in CKD cases. WNV RNA and specific immunoglobulins were detected in 7 (5.7%) and 5 (4.1%) individuals, respectively. The AKI patients with WNV RNA in blood and urine had underlying diseases requiring immunosuppressive therapy and demonstrated moderate to high viral loads. No clinical symptom related to WNV infection were observed in CKD cases with detectable viral nucleic acids. All WNV sequences were characterized as lineage 1 clade 1a and several amino acid substitutions with unknown impact were noted. Detailed epidemiologic investigation of WNV RNA positive CKD cases revealed probable vector-borne virus exposure, without the evidence for transmission via hemodialysis., (© 2015 Wiley Periodicals, Inc.)

Background: Toscana virus (TOSV) is a sandfly-borne pathogen causing febrile diseases and neuroinvasive infections in humans. Definitive diagnosis of TOSV infections frequently requires the detection of viral RNA in cerebrospinal fluid (CSF) or in circulation, which can be achieved prior to seroconversion., Objectives: To evaluate TOSV excretion in urine and impact of urine as a diagnostic specimen., Study Design: A total of 82 plasma, CSF and urine samples were collected from 24 individuals with a preliminary diagnosis of atypical viral encephalitis, where frequent bacterial fungal and viral causes were ruled out. Phlebovirus and WNV nucleic acids were investigated via real-time and nested polymerase chain reaction (PCR) assays. Commercial immunofluorescence assays were employed for viral IgM detection. Amplicons were characterized via cloning and sequencing., Results: Phlebovirus PCR yielded positive results in 7 out of 14 samples that comprise 4 plasma and 3 urine specimens from 3 individuals. Amplicons were characterized as TOSV genotype A. Investigation of the follow-up samples suggested that virus shedding in urine coincides or follows viremia. Despite conserved sequences observed in paired or sequential plasma-urine specimens, L693S substitution in the viral polymerase was characterized in a urine sample., Conclusions: These preliminary findings indicate that urine can be employed as a additional clinical sample for TOSV RNA detection in suspected cases, especially in individuals where specimens for viral diagnostics during the early stages of the infection are not available., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Toscana virus (TOSV), a sandfly-borne phlebovirus, is an important agent of human meningoencephalitis in the Mediterranean region, for which vertebrates acting as reservoirs have not yet been determined. This study investigates TOSV and Leishmania infections in dogs, cats, sheep, and goats from Adana and Mersin provinces in southeastern Turkey. TOSV neutralizing antibodies were demonstrated in 40.4% of the dog and 4% of the goat samples. TOSV RNA was detected in 9.9% of the 252 samples that mainly comprise dogs (96%). Thus, canine species can be suggested as the candidate reservoirs of TOSV. Partial sequences revealed the activity of TOSV genotypes A and B. In two dogs presenting with symptoms of canine leishmaniasis, infections of TOSV genotype B and Leishmania infantum have been documented, describing the first report of coinfections with these agents., (© The American Society of Tropical Medicine and Hygiene.)

Unlabelled: A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies., Importance: Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results, which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We sequenced West Nile viruses (WNVs) from Turkey and found close relationships to WNV lineage 1 strain ArB310/67 from the Central African Republic, distinct from other WNVs circulating in the Mediterranean Basin, eastern Europe, and the Middle East. These findings suggest independent introductions of WNV strains from Africa to the Middle East.

Background: Phlebotomine sandflies are vectors of several pathogens with significant impact for public health. This study was conducted to investigate and characterize phlebovirus and Leishmania infections in vector sandflies collected in the eastern Thrace region in Turkey and Northern Cyprus, where previous data indicate activity of these agents., Methods: Field sampling of sandflies was performed at 4 locations in Edirne and Tekirdag provinces of eastern Thrace and at 17 locations in Lefkosa, Girne, Magosa and Guzelyurt provinces of northern Cyprus. In sandfly pools, phlebovirus RNA and Leishmania DNA were screened via a generic polymerase chain reaction (PCR) and kinetoplast minicircle PCR, respectively. Selected sandfly specimens unsuitable for pathogen detection were identified to species level. Cytochrome oxidase 1 gene region was used for DNA barcoding of selected specimens and pathogen positive pools. Positive amplicons were cloned and characterized by sequencing., Results: A total of 2690 sandflies, collected from Eastern Thrace (15.4%) and Northern Cyprus (84.6%) were evaluated. Morphological examination of 780 specimens from Cyprus exhibited Phlebotomus perfiliewi sensu lato (72.6%), Phlebotomus tobbi (19.7%), Phlebotomus papatasi (2.8%), Laroussius sp. (1.6%) and Sergentomyia azizi (1.6%), Sergentomyia sp. (0.9%), Sergentomyia minuta (0.5%) and Phleobotomus jacusieli (0.1%) species. Pathogen screening was performed in 1910 specimens distributed in 195 pools. In eight pools of P.tobbi sandflies collected in Cyprus, Leishmania infantum DNA was demonstrated. Toscana virus (TOSV) genotype A sequences were identified in two pools of P. perfiliewi s.l. and one pool of P.tobbi sandflies from Cyprus. Co-infection of TOSV and Leishmania infantum was characterized in a P.tobbi pool. Sequences belonging to novel phleboviruses are revealed in three P. perfiliewi s.l. pools. One sequence, provisionally named Edirne virus, identified in Edirne province in eastern Thrace, demonstrated the highest rate of genomic similarity to Adria and Salehabad viruses. Furthermore, Girne 1 and Girne 2 viruses, identified in Girne province, revealed similarities to TOSV and Sandfly Fever Sicilian virus and related strains, respectively., Conclusions: Activity of TOSV genotype A strains in Cyprus and co-infection of sandfly vectors with L. infantum was documented for the first time. Novel phlebovirus strains of unknown medical significance was identified in sampling regions.